Endogenous proteolytic enzymes serve to degrade invading organisms, antigen-antibody complexes and certain tissue proteins which are no longer necessary or useful to the organism. In a normally functioning organism, proteolytic enzymes are produced in a limited quantity and are regulated in part through specific inhibitors.
Metalloproteinases are enzymes present in the body which are often involved in the degradation of connective tissue. While some connective tissue degradation is necessary for normal functioning of an organism, an excess of connective tissue degradation occurs in several disease states and is believed to be attributable, at least in part, to excess metalloproteinase. It is believed that metalloproteinases are at least implicated in periodontal disease, corneal and skin ulcers, rheumatoid arthritis and the spread of cancerous solid tumors.
These diseases generally occur in areas of the body which contain a high proportion of collagen, a particular form of connective tissue. An examination of patients with these diseases of connective tissue has revealed an excessive breakdown of the various components of connective tissues, including collagen proteoglycans and elastin. Therefore, it has been deduced that an excessive concentration of a particular metalloproteinase, for example collagenase, proteoglyconuse, gelatinase, and certain elastases, may cause or exacerbate the connective tissue destruction associated with the aforementioned diseases.
In the normal state, the body possesses metalloproteinase inhibitors which bind to metalloproteinases to effectively prevent these enzymes from acting on their connective tissue substrates. Specifically, in a healthy organism, metalloproteinase inhibitors are present in concentrations sufficient to interact with metalloproteinases to an extent which allows sufficient quantities of metalloproteinase to remain active while binding the excess metalloproteinase so that the connective tissue damage seen in the various diseases does not occur.
It is postulated that one immediate cause of the connective tissue destruction present in the foregoing disease states is an imbalance in the relative metalloproteinase/metalloproteinase inhibitor concentrations. In these situations, either due to an excessive amount of active metalloproteinase or a deficiency in the amount of active metalloproteinase inhibitor, the excess metalloproteinase is believed to cause the connective tissue degradation responsible for causing or exacerbating the disease. It is postulated that, by treating persons with connective tissue diseases with metalloproteinase inhibitors, the degradative action of the excess metalloproteinase may be curtailed or halted. Therefore, particular metalloproteinase inhibitors of specific interest to the present inventors are collagenase inhibitors because it is believed that these inhibitors would be pharmaceutically useful in the treatment or prevention of connective tissue diseases.
The existence of metalloproteinase and metalloproteinase inhibitors has been discussed in the scientific literature. For example, Sellers et al., Biochemical And Biophysical Research Communications 87:581-587 (1979), discusses isolation of rabbit bone collagenase inhibitor. Collagenase inhibitor isolated from human skin fibroblasts is discussed in Stricklin and Welgus, J. B. C. 258:12252-12258 (1983) and Welgus and Stricklin, J. B. C. 258:12259-12264 (1983). The presence of collagenase inhibitors in naturally-occurring body fluids is further discussed in Murphy et al., Biochem. J. 195:167-170 (1981) and Cawston et al., Arthritis and Rheumatism, 27:285 (1984). In addition, metalloproteinase inhibitors are discussed by Reynolds et al. in Cellular Interactions, Dingle and Gordon, eds., (1981). Although these articles characterize particular, isolated metalloproteinase inhibitors and discuss, to some extent, the role or potential role of metalloproteinases in connective tissue disease treatment and speculate on the ability of metalloproteinase inhibitors to counteract this destruction, none of these researchers had previously been able to isolate a portable DNA sequence capable of directing intracellular production of metalloproteinase inhibitors or to create a recombinant-DNA method for the production of these inhibitors.
Surprisingly, the present inventors have discovered a portable DNA sequence capable of directing the recombinant-DNA synthesis of metalloproteinase inhibitors. These metalloproteinase inhibitors are biologically equivalent to those isolated from human skin fibroblast cultures. The metalloproteinase inhibitors of the present invention, prepared by the recombinant-DNA methods set forth herein, will enable increased research into prevention and treatment of metalloproteinase-induced connective tissue diseases. In addition, the metalloproteinase inhibitors of the present invention are useful in neutralizing metalloproleinases, including the excess metalloproteinase associated with disease states. Therefore, it is believed that a cure for these diseases will be developed which will embody, as an active ingredient, the metalloproteinase inhibitors of the present invention. Furthermore, the metalloproteinase inhibitors of the present invention are capable of interacting with their metalloproteinase targets in a manner which allows the development of diagnostic tests for degradative connective tissue diseases using the newly discovered inhibitors.
The recombinant metalloproteinase inhibitors discussed herein interact stoichiometrically (i.e., in a 1:1 ratio) with their metalloproteinase targets. In addition, these metalloproteinase inhibitors are heat resistant, acid stable, glycosylated, and exhibit a high isoelectric point.